Microbial electro-photosynthesis

ABSTRACT

Methods and apparatus for growing photosynthetic organisms lacking Photosystem II (PSII) function using externally supplied electrons shuttled into the organism using redox mediators to improve photosynthetic output and to produce and recover chemicals of interest. By removing PSII, all PAR photons are funneled toward Photosystem I, thereby significantly increasing the theoretical photon utilization efficiency for CO 2  fixation, energy storage and the capacity to synthesize valuable chemicals. Additional genetic modification can be performed to insert or enhance specific metabolic pathways to generate products of commercial interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/972,977, filed on Dec. 17, 2015, which claims priority to U.S.Provisional Patent Application No. 62/093,863, filed Dec. 18, 2014, theentire contents of which are incorporated herein in their entirety byreference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under DE-SC0001016awarded by the Department of Energy. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Over 2 billion year ago, the evolution of oxygenic photosynthesiscompletely transformed the earth's ecosystems by enabling the extractionof electrons and protons from abundant sources of water using sunlightand releasing O₂ into the previously anaerobic atmosphere. Solar-drivenwater oxidation is uniquely performed in nature by the Photosystem II(PSII) complex; however, under normal operation PSII generates and isdamaged by reactive oxygen species, which also damage other cellcomponents. Moreover, the oxygen released during photosynthesis inducesstress to the organism and impairs other important metabolic functions,such as CO₂ fixation and H₂ production. The net output of the lightreactions of photosynthesis is to produce ATP and NADPH, which are usedby the organism to make a wide array of complex organic molecules, manyof which are cost prohibitive to make synthetically for commercialapplications.

SUMMARY OF THE INVENTION

While photosynthetic microorganisms offer a promising route to generatesustainable transportation fuels and petroleum substitutes using solarenergy, boosting their photosynthetic efficiency will help ensure itseconomic viability. A major source of inefficiency is thatphotosynthetically active radiation (PAR, 400-700 nm) constitutes lessthan half of the solar energy reaching the earth's surface.Photosynthetic organisms use PAR to oxidize water as a source ofelectrons in photosynthesis. Photovoltaics (PV) capture twice as manyphotons as photosynthetic pigments do and can be used along withsynthetic catalysts to extract electrons from water. However, artificialsystems have yet to realize the capacity of photosynthesis to producecomplex molecules and drop-in transportation fuels.

Integration of phototrophic and heterotrophic microorganisms intoelectrochemical cells has enabled selection of organisms that donateelectrons to or receive electrons from an electrode, directly or througha mediator, to produce electricity or to promote various metabolicactivities (e.g., biosynthesis, bioremediation). Microbialelectrosynthesis cells (MEC) use microorganisms to synthesize simpleorganic molecules from electrons injected directly from a cathode poisedat significant reducing potentials. However, the required negativepotentials lower the thermodynamic efficiency and may produce undesiredproducts, such as H₂.

Microbial electro-photosynthesis (MEPS) leverages the efficiency ofartificial systems to use sunlight to extract electrons from water andthe capacity of photosynthetic organisms to capture and convert CO₂ intovaluable products and fuels. The electrons are shuttled using redoxmediators from a cathode of an electrochemical cell into aphotobioreactor (PBR) containing a phototroph lacking or having minimalPSII function to promote accepting external electrons. Expanding MECs toinclude photosynthetic organisms leverages the capability of naturalphotosynthesis to use sunlight to boost an electron's reducing potential(more negative) in a controlled manner, where it can be shuttled intothe desired metabolic pathways.

In the MEPS system, a cyanobacterial strain, such as a Synechocystis sp.PCC 6803 mutant lacking PSII, is provided with electrons from anartificial water-oxidation catalyst, which are shuttled into theorganism using chemical redox mediators. By removing PSII, all PARphotons are funneled toward PSI, thereby significantly increasing thetheoretical photon utilization efficiency for CO₂ fixation. The catalystcan be driven directly by light using a photovoltaic (PV) device or fromvirtually any source of electricity in order to leverage ongoingdevelopments in energy technologies. Low-cost electricity-storagetechnologies are required to expand solar installations so that theymake up a significant fraction of total energy production due toimbalance of supply and demand. Chemical mediators are selected anddesigned to reduce naturally occurring electron carriers, such asplastoquinone, within photosynthetic membranes; these electrons are thenbe used by the photosynthetic electron transport chain (PETC) in lieu ofelectrons generated by PSII.

The Synechocystis mutant retains PSI, which uses light energy to boostthe reducing power of these electrons to enable carbon fixation,yielding 1) biomass, 2) complex, high-energy transportation fuelfeedstock, which are not efficiently generated by microorganisms fromelectricity alone, 3) ATP production, and 4) other reduction reactions,such as nitrate reduction.

MEPS has the ability to significantly increase the productivity ofphotosynthetic organisms to make products and fuels, reduce water andnutrient usage, as well as to capture and recycle CO₂. Thus, MEPS helpsaddress the critical domestic and global challenges of energy securityand climate change.

The photobioreactor used with embodiments described herein may beselected from a vertical tubular, air-lift, horizontal tubular,flat-panel, or plastic-bag. Moreover, the anode may include a catalystto promote water oxidation, such as platinum, platinum-carbon, cobaltphosphate, C_(O3)O₄ nanoparticles, and C_(O2)O₃ nanoparticles. Thecathode may be made of carbon felt or carbon fiber. And the phototrophicorganism may be a mutant cyanobacterium Synechocystis sp. PCC 6803 withno or minimal PSII function, a mutant of any species of cyanobacteriumor alga lacking or with minimal PSII function (i.e., 10% or less of wildtype activity), or any species of green sulfur bacterium, greennon-sulfur bacterium, purple sulfur bacterium, purple non-sulfurbacterium or heliobacterium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of microbial electro-photosynthesis (MEPS).PV-driven water oxidation generates electrons and protons to reducechemical mediators. The mediators provide electrons to thephotosynthetic electron transport chain to enable light-driven CO₂fixation and growth of a photosynthetic microorganism lacking PSIIfunction. In some embodiments, PV may be placed beneath the PBR tocollect unused, transmitted light, which is predominantly composed ofinfrared light.

FIG. 2 illustrates a modified photosynthetic electron transport chain.The schematic shows how redox mediators provide electrons (and protons)to the photosynthetic electron transport chain (PETC) of the PSII-lessphotosynthetic microorganism. The mediator provides both electrons andprotons to reduce plastoquinone to plastoquinol or directly transferreducing equivalents to cytochrome b₆f. The electrons receive a boost inenergy from PSI to enable CO₂ fixation, ATP production, and productionof a desired product or biomass.

FIG. 3 shows a plastoquinone variant with a four-carbon alkyl tail (C4).

FIG. 4 depicts a high-surface-area cathode (left) and theelectrochemical cell (right), where the high-surface-area cathodereduces the chemical mediator, the membrane allows passage of protonsfrom the anode, and the platinum-carbon anode catalyzes water oxidation.

FIG. 5 illustrates light saturation in photosynthesis at modest lightintensities. MEPS has the potential to capture and convert many morephotons than natural photosynthesis under moderate to high lightintensities.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments, a strain of cyanobacteria, algae, or any otherphotosynthetic microorganism that retains Photosystem I, but lacks thePhotosystem II function, is used to synthesize fuels and/or productswith electrons provided from an external water oxidation catalyst ratherthan photosynthetic water splitting.

In other embodiments, directed evolution of a photosynthetic organismthat retains Photosystem I, but lacks the Photosystem II function, isused to further boost productivity of photosynthetically generated fuelsand chemicals using electricity. In still other embodiments, light andelectricity assisted growth and synthesis of fuels and products areutilized.

In further embodiments, customized quinone-type mediators for injectingelectrons into the photosynthetic electron transport chain are utilized,for example, plastoquinone variants are used for shuttling electronsinto organisms, as shown in FIG. 3, where is the native isoprenoid tailof PQ is replaced with a synthetically simpler alkyl tail of variouslengths (e.g., 2 to 9 carbons) to adjust the requirements for solubilityand interfacing with native electron carriers.

According to an embodiment, strains of cyanobacteria, algae, or otherphototrophs lacking the PSII function take up externally suppliedelectrons. For example, in the MEPS system, a mutant of thecyanobacterium Synechocystis sp. PCC 6803 lacking PSII function isprovided with electrons from an artificial water-oxidation catalyst,which are shuttled into the organism using chemical redox mediators.

TABLE 1 List of chemical mediators and potential natural electronacceptors, with the corresponding midpoint potentials (E⁰,) and numberof electrons transported (# e⁻). Name E⁰ , mV # e− Prospective RedoxMediators Duroquinone 5 2 Plastoquinone variant 80 2 Benzoquinone 280 2Methylene Blue 11 2 Thionine 64 2 Natural Electron AcceptorsPlastoquinone 80 2 Cytochrome b₆f >80 2

A system to perform MEPS is shown in FIG. 1. PV devices drive asynthetic catalyst to oxidize water (in place of PSII), and theextracted electrons and protons are used to reduce chemical redoxmediators, such as those in Table 1 above. Oxidation of variousmediators by the cyanobacteria is gauged by light-dependent currentproduction and proportional activity of the photosynthetic electrontransport chain.

The reduced mediators are pumped into a vertical tubular photobioreactor(PBR) for utilization by the cyanobacteria and oxidized mediators pumpedout of the bottom of the PBR through a highly porous cathode forregenerating the mediators. A wide variety of existing PBR designs(e.g., horizontal tubular, airlift, flat panel, plastic-bag, etc.) couldalso be used.

The cathode is made of high-surface-area carbon materials, such ascarbon felt or carbon fibers, to maximize the volumetric current densityof the electrochemical cell, as shown in FIG. 4. The cathode is <2 mmthick to minimize Ohmic losses. The electrode unit can be placedunderneath the PBR unit or located in a centralized mediatorregeneration facility. The anode consists of a platinum—carbon (Pt/C)electrode to oxidize water as a source of electrons, or any otherwater-oxidation catalyst, such as cobalt-phosphate and C_(O3)O₄ orC_(O2)O₃ nanoparticles. The anode and cathode are pressed intomembrane/electrode assemblies that use proton exchange membranes (PEM)to separate the electrodes, as shown in FIG. 4.

Small quantities of oxygen are required to enable chlorophyllbiosynthesis and respiration at night for sustained growth. In someembodiments, O₂ will be delivered at night or with the cathode potentialturned off while O₂ is present to minimize mediator-catalyzed oxygenradical formation and current leading to O₂ reduction.

Non-Limiting Examples

We have engineered a strain of Synechocystis that lacks PSII functionand is capable of photoheterotrophic growth, but not photoautotrophicgrowth and thus evolves no oxygen. Therefore, in the presence of light,but without organic carbon, these mutants accept electrons from theprovided mediators to enable photoelectroautotrophic growth and CO₂fixation. The growth rate of the mutant Synechocystis within MEPS isfurther improved using selective pressure for photoelectroautotrophicgrowth (i.e., growth due to both electric potential and light) byremoving the supplied organic carbon (e.g., glucose), but retaining thepresence of inorganic carbon (e.g., CO₂ or bicarbonate), reducedchemical mediators, and light. In some embodiments, the rate ofimprovement may be accelerated using mutagens, such as ultraviolet lightand nitrosoguanidine. In other embodiments the cells will be maintainedin the exponential growth phase by dilution with fresh media.

Strains with good photoelectroautotrophic growth characteristics arefurther modified to enhance their potential to produce products or fuelprecursors, such as by means of free fatty acid production and excretionto reduce harvesting costs. MEPS systems may use platinum electrodes tooxidize water, or a number of other advanced catalysts available on themarket or under development. MEPS replaces electrons provided by PSIIwith those provided externally as delivered by redox mediators. FIG. 2shows how the mediators intercept the PETC of the mutant Synechocystis.While a number of redox mediator types may be used, quinone-type redoxmediators with midpoint potentials near 0 mV may reduce plastoquinonesor directly bind to cytochrome b₆f to deliver its electrons and protons,thus, activating cytochrome b₆f to pump protons for making ATP.

Advantages Over Current Technologies

MEPS provides the following improvements to existing technologies thatuse photosynthesis: 1) PV absorbs photons further into the infrared(700-1100 nm) than photosynthetic pigments (less than 700 nm), allowingfor capturing about twice the number of photons compared to capture byphotosynthetic microbes alone; the combined bio +PV system could haveefficiencies >25%, or a >5X increase over bio alone. 2) Without PSII,PSI receives up to twice as many PAR photons, which would increase thefraction of photons converted to electrons for CO₂ fixation by another30%, after accounting for additional cyclic electron transfer driven byPSI for producing ATP without the protons generated by PSII. Together 1and 2 make significant improvements in utilizing the light energy forphotosynthesis. 3) In place of PSII, MEPS establishes and regenerates alarge pool of reduced chemical redox mediators that allow electrons tobe taken up by the organism on demand. 4) Removing PSII fromSynechocystis eliminates oxygen production, thus reducing the overalloxidative stress to the organism; also, PSII photoinhibition and repairno longer occur. 5) Oxygen-sensitive enzymes like hydrogenase, which arenormally active only at low light levels, could be active throughout theday for producing fuel. 6) MEPS produces biomass and transportation fuelprecursors, both of which are not efficiently generated from electricityalone.

Unlike traditional electrofuels, which make simple fuels fromelectricity, MEPS uses light energy to synthesize complex fuels andproducts with higher energy density that are compatible with existinginfrastructure. Genetic engineering was used to remove PSII fromSynechocystis and could allow for additional improvements tophotosynthesis. Furthermore, new pathways could be introduced forincreased biofuel production and excretion, converting our MEPS systeminto a microbial biofuel factory. The modular MEPS design allows forinterchanging components (e.g., PBR, catalyst, source of electrons) andbe driven by electricity from a variety of sources to leverage otherdevelopments in energy production. For example, transformation ofSynechocystis sp. PCC 6803 with the thioesterase (fatB) gene fromUmbellularia californica to produce laurate, and the native slr1609 genecoding for the acyl-ACP synthetase is deleted, which leads to a lack oflaurate reincorporation and thereby excretion.

Current photosynthesis-based technologies rely on PSI and PSII forcapturing PAR to drive carbon fixation. MEPS utilizes PV to oxidizewater and to provide electrons to photosynthetic organisms in place ofPSII; this represents a significant and innovative departure fromconventional bioenergy technologies. With only PSI utilizing PARphotons, MEPS has the potential to increase the flux of thephotosynthetic electron transport chain.

Electricity from PV does also need light, but can use IR (infrared)photons that are not useful for driving cyanobacterial photosynthesis.If the PV is placed below a thin PBR in a tandem design, it can absorbthe IR photons passing through the PBR, which are not absorbed innatural photosynthesis (FIG. 1). These photons are about as numerous asPAR photons, and high-efficiency PV panels “shaded” by thin PBRs canprovide about as many electrons as are needed for linear photosyntheticelectron transport to NADP and CO₂ fixation.

Under full sunlight, about 90% of the PAR photons are wasted duringnatural photosynthesis. The loss of energy is a regulatory responseprimarily to protect the cell from reactive oxygen species (ROS) underlight stress that can lead to cell death. By removing PSII and thecorresponding light-dependent O₂ production, MEPS strains significantlyreduce oxidative stress and eliminate the major need for the cell todissipate this energy under high light. Without this constraint in MEPS,it may be possible to further streamline the photosynthetic regulatoryprocess so that PSI can utilize significantly more photons for ATPsynthesis and CO₂ fixation to catalyze additional growth at high lightintensity, as shown in FIG. 5. Oxygen-sensitive enzymes likehydrogenase, which are normally active only at low light levels, couldbe active throughout the day and available to produce fuel. Furthermore,growing cultures under conditions associated with MEPS facilitates theselection of strains that best respond to using external electrons andare productive at low oxygen levels. Over time, natural selectionexploits the advantages of photosynthesis at low oxygen levels in orderto utilize electrons from artificial systems and correspondinglyincrease productivity.

Photosynthetic microorganisms have the capacity to produce significantquantities of transportation fuels while using marginal lands that donot compete with agricultural crops. Significantly increasing fuelproductivity correspondingly reduces the water required per gallon offuel. Synechocystis can also be grown using seawater to reducefreshwater needs.

Further enhancements may also be possible, by incorporating genes fromother organisms to synthesize or overexpress other valuable bioproducts.Integrating the genetic changes of the Synechocystis evolved for growingon mediators into fatty acid excreting strains of Synechocystis allowsfor continuous production and harvesting of biofuels as a microbialfactory. We currently have a strain of Synechocystis for excreting thehigh quality feedstock of laurate (C12), which is readily converted tosynthetic paraffinic kerosene (SPK) by decarboxylation and isomerizationusing a catalyst at low cost. SPK is compatible with conventionalgasoline engines and transportation infrastructure, and can be used inaircraft when blended with jet fuel (50% v/v). Together, thesemodifications demonstrate how critical it is to have a versatile genetictoolkit for optimizing biofuel and bioproduct production to ensurecommercial viability.

The modularity of the MEPS design allows the various components (e.g.,PBR, electrodes, catalyst, pumps, source of electrons and electricity)to be engineered and optimized independently, so they can be replacedwith off-the-shelf components when possible. For example, the main PBRchamber could be replaced with many available designs, such asflat-panel, air-lift or plastic-bag PBRs. In this way, the rapiddevelopment of higher performance and lower cost components can beeasily integrated to quickly improve overall economic viability.Similarly, the described MEPS design does not depend on the source ofelectrons provided to reduce the mediators. Anode respiring bacteria(ARB) are a class of bacteria that are able to deposit “spent” electronson an anode from biodegradable organic compounds or biomass, often fromwastewater. Mixed cultures of ARBs and non-ARBs, such as fermenters andmethanogens, can work syntrophically to extract electrons from organicsubstrates and transfer them efficiently to the anode. These substratescould also come from photosynthetic organisms. Regardless of the sourceof electrons, they receive a boost in potential from a PV cell to reducethe mediators for MEPS.

A major advantage of the MEPS system is in its operational flexibility.Co-localizing PV with the MEPS helps to synchronize the energy inputsand keep photocurrent production in phase with biomass production. Inaddition, since photosynthesis only absorbs light up to 700 nm, PV couldbe installed beneath the PBRs to utilize the 700-1100 nm light to drivewater oxidation (FIG. 1).

The current generated by the PV in a tandem cell design is adequate todrive MEPS without any additional land. Our modular MEPS design couldalso accept electricity from virtually any source. Utilizing grid powerallows the PV to be installed at locations that may be less desirablefor MEPS systems, such as residential or commercial rooftops and aridlands without access to water. In addition to PV, grid power can also beprovided to MEPS by other renewable sources such as wind, geothermal,and hydroelectric power; MEPS allows for using this energy to directlymake renewable fuels.

Operational costs for MEPS systems could also be reduced by co-locatingwith power plants, to utilize waste heat, water and CO₂ as well aselectricity to drive the MEPS systems. Thus, MEPS could providebioremediation capabilities by capturing and recycling waste CO₂ andprovide an indirect route for converting hydrocarbons like natural gasand coal to high-value liquid transportation fuels.

The following claims are not intended to be limited by the examples andembodiments described above.

What is claimed is:
 1. A microbial electrosynthesis cell apparatuscomprising: a photobioreactor; a liquid disposed within thephotobioreactor containing a microbe, wherein the microbe is an algae,comprising a mutation in a gene encoding a protein of Photosystem II(PSII), wherein PSII function in the microbe is minimal or absent; anelectrode unit comprising an anode and a cathode, wherein the electrodeunit is in fluid communication with the photobioreactor; a power sourcecoupled to the electrode unit wherein, a source of electrons isgenerated in the electrode unit; and a redox mediator for shuttlingelectrons between the microbe and the cathode.
 2. The apparatus of claim1 wherein the photobioreactor is selected from the group consisting of avertical tubular, air-lift, horizontal tubular, flat-panel, orplastic-bag photobioreactor.
 3. The apparatus of claim 1 wherein theanode further comprises a catalyst to promote water oxidation togenerate the electrons.
 4. The apparatus of claim 3, wherein thecatalyst is selected from the group consisting of platinum,platinum-carbon, cobalt phosphate, C_(O3)O₄nanoparticles, and C_(O2)O₃nanoparticles.
 5. The apparatus of claim 1 wherein the cathode furthercomprises carbon felt or carbon fiber.
 6. The apparatus of claim 1,wherein the power source is a photovoltaic device.
 7. The apparatus ofclaim 1, wherein the source of electrons is provided by one or more ofthe group consisting of water oxidation, water electrolysis, hydrogengas, hydrogen sulfide, the breakdown of organic waste, metal corrosion,cultures of anode respiring bacteria (ARB) and non-ARBs such asfermenters and methanogens that syntrophically breakdown organic waste.8. The apparatus of claim 1, wherein the electrons are shuttled to themicrobe using a redox mediator selected from the group consisting ofduroquinone, trimethylquinone, 2,5 dimethylquinone, 2,6 dimethylquinone,2,3 dimethylquinone, benzoquinone, 2,6-di-tert-butyl-1,4-benzoquinone,or ubiquinone.
 9. The apparatus of claim 1, wherein the externalelectrons are shuttled to the microbe using a redox mediator selectedfrom the group consisting of a plastoquinone (PQ) variant, where thenative isoprenoid tail of PQ is replaced with an alkyl tail of 2, 3, 4,5, 6, 7, 8 or 9 carbons.
 10. The apparatus of claim 1, wherein theexternal electrons are shuttled to the microbe using a redox mediatorselected from the group consisting of methylene blue, thionine,rezasurin or a protein redox mediator.
 11. The apparatus of claim 6,wherein the photovoltaic device is located beneath the photobioreactorand configured to produce electricity using the light not absorbed bythe microbe.
 12. The apparatus of claim 1, wherein the electrode unit isconfigured to transfer reduced redox mediators into the photobioreactorand the photobioreactor is configured to transfer oxidized redoxmediators back to the electrode unit.
 13. The apparatus of claim 1,wherein the cathode is porous such that transfer of reduced redoxmediators and oxidized redox mediators through the cathode occurs.